Time-of-flight (TOF) mass spectrometry has the potential for low cost, high sensitivity analysis of biological molecules covering a wide range of masses. Its high sensitivity results from the multiplex recording advantage, that is: the ability to record ions of all masses form each ionization event. When coupled with new ionization methods, such as matrix-assisted laser desorption/ionization (MALDI), the molecular weights of proteins weighing up to 300 K daltons can be determined. At the same time, such instruments generally have low mass resolution, and mass spectra reveal little structural information. In addition, tandem configurations capable of inducing fragmentation and recording product ion mass spectra from mass-selected components of a complex biological mixture have not been available. During the current grant period the applicants have constructed a tandem TOF mass spectrometer using two reflectron mass analyzers. Their target instrument is one that would provide structural (amino acid sequence) information for peptides in the mass range of 300 to 3000 daltons. This enabled them to design a compact, low voltage (4kV) instrument utilizing collision-induced dissociation (CID) for efficient fragmentation. In the double-reflectron mode, mass resolutions up to one part in 13,000 have been obtained for molecular ions. In the tandem mode, they have been able to obtain product ion mass spectra of a number of organic compounds and small peptides. Moreover, in the course of their work, they have developed a non-linear field (curved field) reflectron that enables them to retain multiplex advantage at unit resolution for product ion mass spectra, and obviate the need for scanning the reflectron as has been proposed by other laboratories. Their major focus in the upcoming period is to reduce this technology to practice, through application of the MALDI/RTOF/RTOF to the amino acid sequence analysis of tumor-specific peptides present in complex mixtures of peptide antigens associated with the major histocompatibility complex (MHC). Additionally, they will develop and investigate an electrospray ionization (ESI) source for the tandem instrument, and construct a separate instrument incorporating a single, coaxial, curved- field reflectron that will be used to obtain amino acid sequences of peptides from metastable and CID spectra, and provide high resolution mass spectra of oligonucleotides desorbed from frozen matrices.